Fig. 1. Characterization of the TC-13 cells. (A) When grown on matrigels, TC13 cells form row-like structures reminiscent of angiogenesis in vitro (–VEGF). When treated with an antibody against VEGF (+VEGF), row formation is inhibited and the cells stay rounded. (B) GATA5 transcripts are present only in differentiated cells. Northern blot analyses using 20 µg of total RNA isolated from undifferentiated (–RA) or differentiated (+RA) TC13 cells were used to detect Gata4 or Gata5 mRNA as described in Materials and Methods. (C) Identification of GATA binding activity. Gel Shift assays were carried out using 5 µg of nuclear extracts and a probe corresponding to the –90 bp GATA element of the BNP promoter as detailed in Materials and Methods. Note that GATA4-containing complexes have a higher mobility than GATA5 complexes. The GATA4 antibody totally supershifted the GATA binding in the undifferentiated cell extracts. GATA5 binding was present only in extracts from RA-treated cells and it was blocked by the GATA5 antibody. GATA4 binding was still detected after RA treatment but GATA5 represented the majority of GATA binding. (D) Control Oct1/2 binding using the same extracts as C. (E) Immunocytochemical staining of untreated (–RA) and treated (+RA) TC13 cells. Cells were fixed in methanol and incubation with the different antibodies was carried out overnight at 4°C as described in Materials and Methods. Staining was revealed by an FITC-avidin D conjugate antibody. Green fluorescent nuclear staining for GATA proteins and cytoplasmic labeling for Von Willebrand factor are observed. Note that only endothelial cells (elongated shape) are positive for GATA5 and Von Willebrand factor.